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dc.contributor.authorSikandar, Asfandyar
dc.contributor.authorLopatniuk, Maria
dc.contributor.authorLuzhetskyy, Andriy
dc.contributor.authorMüller, Rolf
dc.contributor.authorKoehnke, Jesko
dc.date.accessioned2022-04-12T08:14:54Z
dc.date.available2022-04-12T08:14:54Z
dc.date.issued2022-03-09
dc.date.submitted2022-01-12
dc.identifier.citationSikandar A, Lopatniuk M, Luzhetskyy A, Müller R, Koehnke J. Total In Vitro Biosynthesis of the Thioamitide Thioholgamide and Investigation of the Pathway. J Am Chem Soc. 2022 Mar 23;144(11):5136-5144. doi: 10.1021/jacs.2c00402. Epub 2022 Mar 9. PMID: 35263083.en_US
dc.identifier.pmid35263083
dc.identifier.doi10.1021/jacs.2c00402
dc.identifier.urihttp://hdl.handle.net/10033/623172
dc.descriptionThioholgamides are ribosomally synthesized and posttranslationally modified peptides (RiPPs), with potent activity against cancerous cell lines and an unprecedented structure. Despite being one of the most structurally and chemically complex RiPPs, very few biosynthetic steps have been elucidated. Here, we report the complete in vitro reconstitution of the biosynthetic pathway. We demonstrate that thioamidation is the first step and acts as a gatekeeper for downstream processing. Thr dehydration follows thioamidation, and our studies reveal that both these modifications require the formation of protein complexes─ThoH/I and ThoC/D. Harnessing the power of AlphaFold, we deduce that ThoD acts as a lyase and also proposes putative catalytic residues. ThoF catalyzes the oxidative decarboxylation of the terminal Cys, and the subsequent macrocyclization is facilitated by ThoE. This is followed by Ser dehydration, which is also carried out by ThoC/D. ThoG is responsible for histidine bis-N-methylation, which is a prerequisite for His β-hydroxylation─a modification carried out by ThoJ. The last step of the pathway is the removal of the leader peptide by ThoK to afford mature thioholgamide. © 2022 American Chemical Society. All rights reserved.en_US
dc.description.abstractThioholgamides are ribosomally synthesized and posttranslationally modified peptides (RiPPs), with potent activity against cancerous cell lines and an unprecedented structure. Despite being one of the most structurally and chemically complex RiPPs, very few biosynthetic steps have been elucidated. Here, we report the complete in vitro reconstitution of the biosynthetic pathway. We demonstrate that thioamidation is the first step and acts as a gatekeeper for downstream processing. Thr dehydration follows thioamidation, and our studies reveal that both these modifications require the formation of protein complexes─ThoH/I and ThoC/D. Harnessing the power of AlphaFold, we deduce that ThoD acts as a lyase and also proposes putative catalytic residues. ThoF catalyzes the oxidative decarboxylation of the terminal Cys, and the subsequent macrocyclization is facilitated by ThoE. This is followed by Ser dehydration, which is also carried out by ThoC/D. ThoG is responsible for histidine bis-N-methylation, which is a prerequisite for His β-hydroxylation─a modification carried out by ThoJ. The last step of the pathway is the removal of the leader peptide by ThoK to afford mature thioholgamide.en_US
dc.description.sponsorshipDeutsche Forschungsgemeinschaft (DFG): MU 1254/32-1 J.K. thanks the BBSRC for support (BB/V016059/1). R.M. would like to acknowledge DFG (Leibniz Award: MU 1254/32-1).en_US
dc.language.isoenen_US
dc.publisherACS/ American Chemical Societyen_US
dc.relation.urlhttps://pubs.acs.org/doi/10.1021/jacs.2c00402en_US
dc.rightsAttribution-NonCommercial-ShareAlike 4.0 International*
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/*
dc.subjectAntibiotic; Antibiotics; Anticancer; Anticancer activities; Anticancer activity; Antiproliferative; antitumor activity; Apoptosis; Archaea; B; Bacillus; Bacterial; Biochemistry; Biology; biosynthesis; Biosynthesis gene cluster; Biosynthetic pathway; Biotechnology; Cancer; Catalytic mechanism; cell; Cell Line; Cell lines; Cells; chemistry; class III; Cloning; Cysteine; Cysteine protease; Cytotoxic; D-amino acid; Decarboxylase; Decarboxylation; dehydration; Development; downstream processing; enzyme; Enzymes; enzymology; Export; Expression; Family; funding; Future prospects; gene; gene cluster; Genome; Genome mining; Genome-mining; Germany; Heterologous expression; hidden Markov model; Histidine; history; In Vitro; In-Vitro; Infection; Inhibitor; Inhibitors; integration; Lanthipeptide; Lanthipeptides; lantibiotic; learning; Libraries; library; lipopeptide; Lipopeptides; machine; Machine learning; macrocyclization; Macrophage; Macrophage polarization; Mass Spectrometry; Mass-spectrometry; Mechanism; Mechanism of action; Metabolism; Metabolomics; methods; mining; Model; Molecular mechanism; natural product; Natural Products; Nomenclature; Novel; Order of reaction; Pathway engineering; peptide; Peptide Library; Peptide natural products; Peptides; Pharmaceutical research; Pharmacological target; phosphotransferase; polarization; Post-translational modification; post-translational modifications; Potent activity; prediction; Promiscuity; protease; Proteases; Protein; protein structure; Proteins; Recommendation; Reconstitution; research; Residues; ribosomally synthesized and post translationally modified peptide; RiPPs; Selectivity; Sequence; Sequence Homology; Serine; Spectrometry; Streptomyces; Structural basis; structural biology; Structure; Structure prediction; substrate; Substrate Specificity; Substrates; Suzuki; synthetase; thioamide; Thioamides; thioholgamide A; Thiopeptide; thioviridamide; Untargeted metabolomics; utilizationen_US
dc.titleTotal In Vitro Biosynthesis of the Thioamitide Thioholgamide and Investigation of the Pathway.en_US
dc.typeArticleen_US
dc.identifier.eissn1520-5126
dc.contributor.departmentDepartment of Microbial Natural Products (MINS), Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), Department of Pharmacy, Saarland University (UdS), Campus E8.1, Saarbrücken, 66123, GermanyDepartment of Pharmacy, Pharmaceutical Biotechnology, Saarland University, Saarbrücken, 66123, GermanyGerman Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Braunschweig, 38124, GermanyWorkgroup Structural Biology of Biosynthetic Enzymes, HIPS, HZI, UdS, Saarbrücken, 66123, GermanySchool of Chemistry, University of Glasgow, Glasgow, G12 8QQ, United Kingdomen_US
dc.identifier.journalJournal of the American Chemical Societyen_US
dc.source.volume144
dc.source.issue11
dc.source.beginpage5136
dc.source.endpage5144
dc.source.journaltitleJournal of the American Chemical Society
dc.source.countryUnited States


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